Abstract: The Notch signaling pathway plays a critical role in multiple cell fate decision events in metazoans, and recentiy has been implicated in the pathology of cancer in humans. Genetic and biochemical evidence has revealed a large network of modulators that are involved in establishing a precise level of Notch signaling and determining tissue-specific outcomes of pathway activation, from the interaction ofthe Notch receptor with its ligands Delta and Serrate to transcriptional regulation of the Notch target genes. Though much of this knowledge has been obtained using Drosophila as a model organism, it is relevant for understanding Notch function in humans due to a remarkable conservation ofthe fundamental steps in Notch signaling between flies and mammals. Yet, a major obstacle has been an inability to study endogenous signaling events. In this study, we propose to gain insight into the Notch signaling events by using the genetic advances in recombineering and targeted transgene insertions to replace the endogenous Notch and Delta genes with their fluorescent and affinity tagged isoforms. These transgenes will be used for studying the composition of Notch containing signaling complexes and for imaging the process of Notch activation in vivo. Completion of these experiments will establish an in vivo map ofthe Notch interaction network and reveal the dynamics of signaling in living cells. With the above in mind, we will pursue the following specific aims: 1) To generate Drosophila lines containing genomically tagged Notch and Delta, 2) To analyze the Notch interaction network in vivo, and 3) To study the dynamics of Notch signaling in vivo. Moreover, this collaboration will have multiple benefits for the continued professional development of Dr. Veraksa's career. While the two principle investigators have a history of productive collaboration. Dr. Veraksa is eariy in his career as an independent investigator;yet both Veraksa and Artavanis-Tsakonas bring complementary expertise to the proposed project. This collaboration will also significantiy benefit from the Training Core.